PROJECT SUMMARY The delicate balance between proliferation and differentiation of stem/progenitor cells of the salivary gland (SG) must be tightly regulated to ensure proper morphogenesis, homeostasis and regeneration. Alterations to normal SG function, clinically often manifested with hyposalivation, are associated with diseases such as Sjgren?s Syndrome and with ?-irradiation therapy of Head and Neck Squamous Carcinoma patients. Currently, treatment options for hyposalivation and mechanisms for restoring normal SG function remain limited. Therefore, the development of new tools and strategies directed at identifying transcriptional and signaling networks underlying stem/progenitor cell function of the SG with the ultimate goal for use in stem cell regenerative-based therapies and tissue engineering approaches, are critical. Hence, our goal is to examine the transcriptome landscape of the stem/progenitor cells of the SG and identify and characterize the regulatory networks of this cell population. It is well established that ?Np63, plays a critical role in epithelial regenerative function as ?Np63-null animals fail to develop several epithelial-rich organs including the SG. However, our current knowledge of how ?Np63 regulates gene expression in the stem/progenitor cells of the SG is lacking. Thus examining the p63-driven regulatory networks is a key step towards a better understanding of the biology of the SG progenitor cells and in directing new strategies in treating SG dysfunction. To address these knowledge gaps, we will utilize SG stem/progenitor cells obtained from novel ?Np63-GFP transgenic mice to study two major areas of interest. First, we will perform functional assays to compare the abilities of ?Np63- GFPhi and ?Np63-GFPlow SG cells to retain both their progenitor capacity and their ability to differentiate into mature cell lineages in SG organospheres (Aim1A). Furthermore, we will perform in vivo transcriptomic profiling (RNA-seq) to generate the mRNA expression profile in a pure population of ?Np63-GFPhi SG progenitor cells and ?Np63-GFPlow SG cells (Aim 1B). Such in vivo studies are important, since they will identify for the first time the gene expression profile of SG progenitor cells on a broad and dynamic scale. Second, we will perform in vivo ChIP-seq studies to identify key regulatory networks and pathways regulated by ?Np63 in SG cells. Collectively, our approach using a genetically-defined model system and cutting-edge next generation sequencing technology will better elucidate the transcriptomic landscape of SG progenitor cells and shed light on the ?Np63-governed transcriptional regulatory network and signaling pathways. Since such genomic studies are particularly lacking for SG cells, our proposed experiments will likely reveal new biomarkers and important regulators and drivers of stem cell function in this organ. Long term, such knowledge will have clinical and therapeutic implications for human patients who suffer from SG dysfunction diseases.